1. Field of the Invention
The invention relates to RNA molecules having adenosine derivatives incorporated into the 5′ end, and methods and materials for their preparation and use.
2. Description of the Related Art
Coenzymes are a group of small organic molecules with distinct chemical functionalities, which are usually unavailable or insufficient in the side chains of the 20 naturally occurring amino acids. A number of metabolic protein enzymes require coenzymes to act as accessory molecules to provide necessary reactivities. Three common coenzymes, coenzyme A (CoA), nicotinamide adenine dinucleotide (NAD), and flavin adenine dinucleotide (FAD) carry out a variety of acyl group transfer and electron/hydride transfer reactions in metabolism. Although CoA, NAD, and FAD function in very different chemical capacities, they share some surprisingly common structural features: a ribonucleoside adenosine at one end, a chemically functional group (pantetheine, nicotinamide, or riboflavin) at the other, and a pyrophosphate connecting the two groups.
Because the structures and functions of these coenzymes are conserved in all kingdoms, CoA, NAD, and FAD are believed to have existed since the last common ancestor of life on Earth. Furthermore, the fact that all these coenzymes contain an adenosine group, one of the four essential building monomers for RNA, suggests the plausibility of their existence and biological function in a more ancient world. Recently, RNA-catalyzed synthesis of these three coenzymes from their corresponding precursors, phosphopantetheine, nicotinamide mononucleotide (NMN), and flavine mononucleotide (FMN) (4) was demonstrated (Huang, F., et al. Biochemistry 39: 15548-15555 (2000)), providing strong experimental evidence of possible coenzyme synthesis and utilization in the RNA world (Gilbert, W. Nature 319: 618 (1986)).
Currently known ribozymes, either naturally occurring or isolated in vitro, however, do not these coenzymes to perform chemistries as protein enzymes do. A recent report has demonstrated RNA-catalyzed acyl CoA synthesis (Jadhav, V. R., and Yarus, M. Biochemistry 41: 723-729 (2002)), and ribozymes have been isolated capable of catalyzing thioester formation (Coleman, T. M., and Huang, F. Chem. Biol., 9: 1227-1236 (2002)). Yet, RNA catalytic activities involving NAD and FAD redox chemistry have not been shown to be within the functional capacity of RNA. Since the development of in vitro selection (SELEX) (Ellington, A. D., and Szostak, J. W. Nature 346, 818-822 (1990); Tuerk, C., and Gold, L. Science 249: 505-510 (1990); Robertson, D. L., and Joyce, G. F. Nature 344: 467-468 (1990)), numerous artificial ribozymes have been isolated from random RNA libraries (Jaschke, A. Curr. Opin. Struct. Biol 11: 321-326 (2001)).
Convenient and efficient in vitro transcription methods have played an important role in the advancement of RNA research by providing easily available RNA with defined sequences. However, all the current methods require a guanosine derivative as the transcription initiator under T3, T7, and SP6 promoters (Milligan, J. F., et al., Nucleic Acids Res. 15: 8783-8798 (1987); Pokrovskaya, I. D., and Gurevich, V. V., Anal. Biochem. 220: 420-423 (1994)). Although other nucleotides such as adenosine and cytidine derivatives have been shown to be able to initiate transcription with certain sequences (Nam, S. C., and Kang, C. W., J. Biol. Chem. 263: 18123-18127 (1988); Helm, M., et al., RNA 5: 618-621 (1999)), the initiation sites are not well defined. The yields are low (Nam, S. C., and Kang, C. W., J. Biol. Chem. 263: 18123-18127 (1988)), and they usually result from non-templated incorporation or omission of one or two nucleotides (Helm, M., et al., RNA 5: 618-621 (1999)).
Development of methods for the preparation of RNA molecules linked to coenzymes and other adenosine derivatives may provide interesting catalysts that have improved or novel properties as compared to current RNA catalysts. Incorporation of fluorophores or biotin at the specific 5′ end of RNA results in site-specifically labeled RNA that may be used in RNA structural and functional investigation and nucleic acid detection.